JPS60120990A - Cloned dna coding rabbit cancer-necrosing factor - Google Patents

Abstract

PURPOSE:To provide the titled cloned DNA which is very useful as a probe or primer in the cloning of a factor nocrosing cancer in different animals including human and is used to form a microorganism or cells for producing the necrosing factor. CONSTITUTION:The DNA which is represented by the formula and has base sequence that can code the polypeptide containing the aminoacid sequence of the factor necrosing rabbit cancer. At first, the cancer-necrosing factor produced in the rabbit body is isolated and purified to clarify the chemical properties of the protein, then the mRNA of the factor is efficiently produced in vitro using macrophage of rabbit albeoruses and the product is isolated and concentrated. Then, gene recombination is effected to clone cDNA coding the factor necrosing rabbit cancer.

Description

[Detailed description of the invention]

The present invention is a cloned I) encoding Utagi necrosis factor.
N.A. The present invention relates to vector DNAs incorporating these vectors and hosts transformed with these vector DNAΔs. The following abbreviations are used herein to simplify the description. A adenino C tonton / G guanine T pumi / Ala arani / ArIr argini / Asn asparagi / Asp asbashiginic acid Cys cydine Gin glutamine G]u glutamic acid GIy glycy / 11is histidi 7 11e isostin / Le11 mouth in / 1. ．． ys Lysine MeL Methyl Nino P amount) C Phenylalanino Pro Pro/ Ser Serino Thr Thre A Nin Trp} Rib l F7 N Tyr Chi 11 N No Va. I valine 1) NA deoxyri I: +A 100 cl) NA complementary DNA 1gNA ribomo66mRNA messenger RNA dAT11 de]quinadenosi/triphosphate dCTPde Phosphate dTTP7'λ
Kisijimiji/Triphosphate Rigo (dC) Oligodeoxy7disyl Rigo (dG
) , 1′ri(d
G);l! Lydeoxy7guanylic acid poly(dT) polypropylene Axidymicylic acid AT+'ade/ene triacetate/acid 11) TA ethylenediami/tetraacetic acid kb4-. jJ ground 1. L kl1p4'Kuchio JI (vs. Carswall et al.
+fn of mice infected with Almette-Guerin (IICG') and then treated with
We found that the serum contained a substance that causes necrosis of the transplanted Matl1^sarcoma, and we named this substance necrosis factor [Proc. Nat. Acad.
Sci. USA, ``72.3G (i (i (1975) Ko. Cancer necrosis prisoner is Makurov 7-') live yI!
It is called an active substance, and its characteristics include (1)
) The cancer in a persimmon becomes necrotic when thrown at a tumor-bearing animal.
+2 reduction (ii) in vitro certain thickness 4
111 cells (for example, mouse cancer cells ■, cells jj, +
, h}% (r'c-to cells from 4 mountains) or have almost no harmful effect on normal cells, and (ti+) the effect is not species-specific. or known. Because of these characteristics, cancer necrosis factor is highly anticipated for its clinical application as a new type of anti-diagnosis agent. However, the conventional method for producing cancer necrosis cells has been applied to animals such as mice and rabbits.
(・t(jir+umacncs injected, then -r7
1 t. It is difficult to stably obtain a large amount of cancer necrosis at a low cost since the cancer necrosis is purified from the blood and body dk after administering the drug. It was the situation. In order to solve the problem of
Although it is conceivable that this technique could be applied to a new technology, the basic knowledge regarding the amino acid sequence of the cancer necrosis factor and the method for collecting the mRNA necessary for its implementation has not been thoroughly unified so far.Therefore, the present invention They first investigated the ρ produced in the rabbit's body.
: Purification of cancer necrosis factor, elucidation of its protein chemical P1 substance, and subsequent induction of efficient production of cancer necrosis factor mRNA in vitro using rabbit alveolar macrophages. I saw 4 and it was a win! Local necrosis factor mRNA was purified and concentrated. Furthermore, by applying the Shoden f'-silk modification technique, rabbit cancer necrosis factor: 'I
The present invention was completed by successfully cloning the Dozuru cDNA. The present invention is based on Uri. 1! Contains the amino acid sequence of cancer necrosis factor and has a base sequence calling J' lipebutite 1)
It provides DNA having NΔ, particularly the following base sequence (r). The DNA shown in item 11 (1) encodes voribebutide having the following amino acid sequence (n). 1.1) N8 (1) may have the following 1111 sequence (II+) at its 5' end. Furthermore. DNA (1) and part 5
I) NA [hereinafter referred to as DNA (1±1)], which has an 11J base sequence indicated by (I'll) at the 5' end, may have ATG (7) at the 5' end. 1) NA DNA having the Kawagoe sequence indicated by the symbol (rl+) at the 5' end of (I') is polypeptide (II).
In addition to (II), the following amino acid sequence (I
V) and the following polypeptide (TV+
I+)] is coded. DNA having the base sequence indicated by ATG at the 5' end of DNA (I) encodes a polypeptide containing Met at the N-terminus of (It) in addition to polypeptide (TI);
) has the base sequence indicated by ATG at the 5' end of
A, polybebutide (rV+rl) is added (IV+
n) encodes a polypeptide with Met at the N-terminus. The present invention includes the above DNA, vector 1 incorporating them.
) Not only hosts transformed with NA and their vector DNAs, but also cases where the above 1') NA contains allelic genetic mutations, degeneracy of the genetic coat, or some modifications,
As long as the polypeptide produced by the host transformed with the vector DNA incorporating them is a polypeptide that exhibits an immunological or biological profile V1 equivalent to Urigi cancer necrosis factor, the above-mentioned DNA modifications ( This also includes cells, vector DNAs into which they have been integrated, and hosts transformed with these vector DNAs.Incidentally, cases in which a portion of the DNA is modified include cases in which a portion of the DNA is missing. The present invention will be explained in detail below. ni, Uyugi ni I'ropioni
Rabbit cancer necrosis factor was produced in the body by administering -bactartumacnes and totoxin, and rabbit cancer necrosis factor was extracted from the blood by a combination of ion exchange chromatography, gel σ3 filtration, etc. Isolated and purified. The rabbit cancer necrosis factor was identified using its biological activity as an index. The index used was (+) mouse LM cell <ATCCCCI. 1.2) and (ii) anti-ulcer activity against Math^sarcoma transplanted into mice. These test methods are described, for example, in JP-A-58-174330. The molecular weight of isolated and purified rabbit cancer necrosis was determined in the presence of 8M urea and in the presence of 8M urea.
! The molecular weight of cancer necrosis derived from rabbit plasma was approximately 45,000 Daltons in the absence of urea.
In the presence of urinary chorda, it dissociates into a single polypeptide, whose molecular weight is approximately 1. [F-10,000 Dalton was arrested. The molecular weight of the complete IlII state is said to be the molecular weight of the basic constituent unit of rabbit cancer necrosis factor. Furthermore,
Partial amino acid sequences including the N-terminus and C-terminus were resolved by the etomanolysis method, the hydrazidegradation method, and the enzymatic method using carboxybeptidase, respectively. As a result, N
It was revealed that the amino acid sequences of the terminal and C-terminal portions have the structure shown below. N-terminus: Ser18la-Ser-＾rg-＾1a...
...CA. ．． End=Val 1-yr-Phe-Gl
y-1lc-Ile-8la-1. Preparation of eu(2) Wooligi cancer necrosis factor mRNA Wooligi cancer necrosis factor mRNA
To get it, first get annoying! I'ropionibacus
A reticuloendothelial system activator such as Tcriullacanes, BcG, or Zymosan is injected intravenously or intraperitoneally. Imitation 7-14 [After 1, slaughter the Urigi and extract the alveoli, peritoneal cavity, blood, and other tissues from III! - get the di. Cultivate this macroph 7-') R}: about 2 x 10 per cJ surface
4-IX 106 seeds sown, 35-38°C1, preferably about 37°C1, in air containing about 5-10% carbon dioxide, humidity about 9
Incubate at 0-100% for about 30 minutes to 2 hours. Next, en-1 toxins obtained from Gram-negative bacteria, such as Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium, bobolita socharide, and a protein synthesis inhibitor (such as cyclohenamide) are added, and the culture is further incubated for 3 to 8 hours. Continue to treat Wooligi cancer necrosis prisoner mRN
A synthesis is induced and accumulated. Note that the pre-culture may be omitted. The amount of addition is generally about 01~+00
Qlzg/ml (final IO degree, same hereinafter), preferably about 1 to 100 μg/ml. At this time,; 1 rubole-12 to myristate-13-acetate, phorbol-12.13-didecanoate, 1, rubole-12
．． About 1-2000 ng/ml of 1, rubole esters, such as 13-dibenzoate, may be added. The amount of the protein synthesis inhibitor added varies depending on the compound i:1 class, etc., but for example, the amount of protein synthesis inhibitor added is
~50llg/+1. As I〆9Jlji,
Various types of synthetic Ig substrates with a Δ value of 1,000 ml are used for animal cell culture, such as Ig's Ml-1640. For example, in ``Cell Culture Manual J,'' edited by Kosuke Munemura, published in 1982, published in 1982, J.
It is preferable to add 1 ri of animal disintegration (for example, 1 ri of fetal bovine blood, calf serum). After the culture is completed, the cells are extracted using a conventional method, such as the method of Cbirgwin et al.
, +8, 5294 (1979)], and then extracted with Alig (dT) cellulose or poly(U) cef7-lose (F7 Leman Ane) according to the 1-pa method.
ftI to adsorption column column using
The poly(^) mRNA fraction is separated by a batch method. This po+y(A) mRNA fraction was subjected to acid urea agarose gel electrophoresis or chromatography. Rabbit cancer necrosis factor mRNA can be purified to a high concentration without being subjected to sugar density gradient centrifugation. mR
It is sufficient to translate it into NA8 protein and examine its biological lfi property. For example, Afri hnomega. r-ru (Xenopus
The mRNA is injected or added to an appropriate protein synthesis system such as mouse I. laevis oocytes, reticulocyte lysate, or wheat germ, and translated into protein. ，
This is done by confirming that it exhibits cytotoxic activity 1'1 against -929 cells and that this cytotoxic activity is neutralized by the antibody against the purified rabbit tumor necrosis factor isolated from rabbit plasma as described above. . Note that the method using African 7-mega frog oocytes is carried out, for example, as follows. Approximately 50 ng of mRNA per oocyte was collected in microorganisms (7') Ethane, ? / method, and 10 of them are injected into 1
00 μl of Perth culture [Gurdon, J. , J.
Embryol. Exp. Morphol. ,20,4
01 (+9 (i8)) at 22°C for 24 hours,
1. After modulation, the centrifuged supernatant (10,000
0rpI1, 10 minutes) as a specimen, and cancer necrosis cell activity is measured using L-920 cytotoxic activity as an indicator [L-
The method for measuring 029 cytotoxic activity V1 is described by Ruff, M. R. ，
etal. , J. Immunol. ,+25,1[17
1 (+980)]. The mRNA encoding the rabbit cancer necrosis factor of the present invention is characterized by the following properties. ■I. It has a size of C ~ 2.7 kb. (2) It has a boriadenylic acid structure at the 3' end. ■Encodes the polypeptide of rabbit cancer necrosis factor. (331 Cloning of necrosis prisoner cDNA from Shiinagi cancer (2)
) was used as a template and oligo (d
dATp, dGTP, dcTr using a synthetic oligonucleotide having a base sequence thought to correspond to the partial amino acid sequence of rabbit cancer necrosis factor or rabbit cancer necrosis factor as a primer.
', Single-stranded cDNA complementary to mRNA is synthesized using reverse transcriptase (e.g., avian myeloid leukemia virus Yamamai reverse transcriptase) in the presence of dTTP, and template mRNA is synthesized by alkaline treatment.
Decompose and remove A. Next, using this I11 chain cl)NA as a template, double-stranded cDNA was generated using reverse transcriptase or Escherichia coli DNA polymerase (large fragment).
Synthesize. I)NA obtained here is converted into poly(c+6
) one poly(dc) or poly(dA)-poly(dT)++
, Mopolymer extension method [Noda, M. , etat. ,N
aLure. 295,202(+982>,Nelso
n, T. S. , “Methods in Enzy*olo
gy″.08,4+(+979>,Acade+icI
ゝr'ess Inc. ．． For example, it is inserted into the restriction enzyme PstI cleavage site of plasmid f) 13R322 according to a conventional method such as [New York]. The obtained recombinant plasmid is processed, for example, by the method of Cohen et al. [Proc. Na
t. ＾cad. Sci. USA, 09, 2+10 (+9
72)], for example, E. A cl) NA library is prepared by introducing the host protein into a host such as the cocoon Chi1776 strain and transforming it, and selecting a tetracyclino-resistant strain. Colony hybridization/testing using the plus/minus method for cDNA libraries with
Anahan, D. , etal. ,Gene,In,0
3 (1980)] to screen the desired clone. That is, using the rabbit cancer necrosis mRNA obtained in step (2) as a template, 32p-labeled cDNA is synthesized and used as an induction plus probe. Separately, using alveolar macrophages obtained from normal, untreated pigs as material, the same procedure 1 was performed except for the induction procedure of cancer necrosis factor mRNA, and the extracted and concentrated mRNA was used as a template to generate 3211-labeled cDNA.
Synthesize A and induce it. From the above-mentioned cDNA library, Jg plus
Select Claw 7, which strongly couples with the blow []-bu and hardly binds with the induced negative blow. Toko 911
Plasmid DNA is isolated from the isolated clone, converted into single-stranded cDNA by heating or alkaline denaturation, and fixed on a cellulose filter. After adding an mRNA fraction containing Ooligi carcinoma necrosis prisoner mRNA to this and hybridizing, the bound mRNA was eluted and recovered, and this was injected into African frog oocytes, and the recovered mRNA
A test is conducted to determine whether the test sample contains rabbit cancer necrosis factor or not (hereinafter referred to as hybridization/translation test). m of rabbit cancer necrosis factor by the above method.
A transformed strain containing a cloned DNA plasmid incorporating a DNA fragment containing a nucleotide sequence complementary to RNA can be obtained. Furthermore, the cloned DNA fragment of this transformed strain was cut out with 6 appropriate restriction enzymes and labeled with 32p, and the DNA fragment was cut out with a 32p restriction enzyme and labeled with 32p.

[By using the above-mentioned cDNA library as a J1j screen, a larger size cl can be obtained]
NA may also be selected. Thus obtained poly(1-I) monomer 1) NΔ
For fragments, for example, the MaxaII-Gilbert method [Proc. Nat. ＾cad. sci. UsA, 74
, 500 (+977)], and searched for the base sequence encoding the partial amino acid sequence (including the N-terminus and C-terminus) of the previously clarified Woori Gi cancer necrosis factor. A base sequence corresponding to the entire translated region of cancer necrosis factor [Shiobuki sequence shown in (1) above]
cl) containing NΔ, a polypeptide containing the amino acid sequence of rabbit cancer necrosis factor was obtained by selecting: +−i sulj*. Cloning 1) NA with Iil+ sequences can be obtained. After the cloned/transformed DNA of the present invention is treated in a sequential manner, it can be incorporated into an appropriate expression vector to obtain a vector for producing necrotic captives from rabbit cancer. As the vector, any vector that grows in the microorganism to be transformed can be used. For example, plasmid (E. coli plasmid, pnR
322, etc.), frugi (Rano-Tafage derivatives, etc.), and viruses (SV40, etc.). these are! 11 alone or in combinations thereof, e.g. pnR322
- It may be in the form of a SV40 hybrid plasmid or the like. I) The site for incorporating NΔ can also be arbitrarily selected. That is, the appropriate morphological position of the expression vector is always
Depending on the person, an appropriate restriction enzyme is applied to cleave it, and the cloned 1) NA is inserted into the cleavage site. It can be processed and incorporated into any length. A vector containing the cloned DNA described above can be transformed by acting on a suitable host by a method to obtain a vector for expressing a trait. Examples include λ vectors such as β-galactondase, tributofinase, β-lactamase, and ArnoJ lyphosphtase.Furthermore, examples of hosts used for transformation include E. coli, Bacillus bacillus, J. Examples include microorganisms such as those mentioned above and cultured cells such as COS monkey cells. 1”
It is extremely useful as a primer or primer, and itself serves as a shrine for identifying cancer necrosis factor-producing microorganisms or cells. The present invention will be described in more detail below with reference to Examples and Reference Examples, but the present invention is not limited to these Examples. Example (1) Isolation and purification of mRNA fraction from rabbit alveolar macrophages. 1! (rest m about 2.5+tg) Propioni
100 dead bacteria bodies per bird
The animals were injected intravenously at a dose of 1.0 mg, and sacrificed 8 days later. Immediately, a thoracic tracheotomy was performed, and alveolar macrophages were collected by repeatedly flushing the lungs with acid-kt-enriched physiological saline through a tube inserted into the trachea. Approximately 3 x 109 Ilj11 macrophages were obtained from 12 rabbits. This III phage was suspended in 1 volume of Rr'MI-1 (i40 medium) containing 10% fetal bovine serum, and 2 x 10 phage per plate was added to Petri dinonyu (ll'l diameter 8c-).
They were sown in 7 pieces and cultured in ni1 culture at 7°C in air containing 5% carbon dioxide and flJICI: 90-100%. After pre-incubation for 1 hour, Entototon (Escherichia coli-derived paste Bobolilinolite'), TPA (phorbol),
12-myristate-13-acetate) and/or

[No. 17 microorganisms were added to the final concentration of 10 μir/Ilt.
IOng/ml and 1μ! The mixture was added and mixed so that the ratio was r/1, and the culture was continued. After 4-4.5 hours (i!!I
At 5 to 55 o'clock II), the culture solution was removed by suction, and Dison,
The remaining Mac [JF7-] from step 1 was dissolved in a 5M guanidyldionanate solution containing 0.6% lithium laupirdinium chloride and OmM lithium citrate; and sonicated. This homogenate was layered on a 5.7 M senium chloride aqueous solution containing OIMEITA, and centrifuged for 20n+f at 20,500 rpm using an ultracentrifuge (RPS27-21, manufactured by Japan ΔY) to remove all RN.
Fraction A was obtained as pellet. Add this to 0.35MNaC
I, 7M containing 20mM Tris and 20mM EDTA
It was dissolved in a small amount of urea solution and recovered as an ethanol precipitate. 5.2 mg of total RNA was obtained from 12 E. chinensis. This total RNA fraction was converted into IOmMTr containing ImMEDTA.
is-IIcIkA8iAM (pll7.4> (hereinafter T
Dissolved 2 ml of the mixture and heated at 65° C. for 5 minutes. After adding NaCI solution'i: 05M to this solution, 1.
314ug of poly(8)mRNAΔ was eluted with 1 + d'ruco and ◆co.
)mRNA? It's τノ. The poly(^)mR obtained here
NANO20 (bzgwoano fu-sgel electrophoresis (gel concentration 1%, (3M urea present 1, pl14) was conducted, and the gel was divided into seven halves according to the understanding (1).
After incubating at 70'C for 10 minutes (1 tl), after extracting with water saturated fume, and extracting with cold water,
Poly(A) was precipitated with Eq.
mRNA was collected. Poly(8) mRNA in each fraction
How to use African frog egg No. 1 cells
At last, the cancer necrosis prisoner mRN'Afit was 11111
and corresponds to a molecular size of 1.6 to 2.7 kb! Rabbit cancer necrosis factor mRNA was collected at a high concentration in the second minute. The specific activity of this purified poly(^) mRNA is approximately 1.23
011'-(:t./μgl SENA, unpurified po
Specific activity of ly(^)mRNA preparation approximately 320 units/tt
I? rQhi was approximately 4 times higher than that of RNA (Figure 1)
. This Wit! k! IJirIPi, which was translated and synthesized using lpoly(8) mRNA as a template and the width of -(-t71J Canoe frog oocytes), is the same as the cancer necrosis factor prepared from rabbit plasma. Confirmed using anti-rabbit cancer necrosis r antibody.
Poly(A) mRNA was injected into the oocytes of γ-Furicanomegae and cultured at 22°C for 24 hours in a birth chamber.
The liquid was used as the sample. Add 20 μl of anti-rabbit islet necrosis factor antiserum manufactured by Kasu to this sample +00Bl, and react at 7°C for 2 hours, then L-02! ) Cytotoxic activity against cells was measured. As a result, in the absence of antibody 1, 1,2f+7
1/1, but in the presence of the antibody, it was 4"//
It became below IN+ and was almost completely neutralized. In Section 2 1, d111 constant viscosity is shown in comparison with the case of Urigi plasma-derived cancer necrosis factor. In the figure, the fist is the African 7-megayl egg [, translated from mRNA in the upper cell].
+1! v. ++ indicates the cytotoxic activity of necrosis prisoner-r1, 0 indicates the cytotoxic activity of Ugigi blood 5π111 Yonejima necrosis factor, dot gland indicates the case of anti-reactive (rI:'h), and the solid line indicates the presence of antibody J1. The following case is shown. The !+'i poly(^) mRNA obtained here was used in the following experiment. (1st generation) (・l) Synthesis of NA Purified poly(^) mRNA 4 μg G was used. cDNA was synthesized as shown in Section 1. Reaction volume: 100 μl 50 mM Tris-11CI buffer (pll8.3):
IOmMMg(12; 70mMKCI; ImM GigiA slay liv; 05mMdTT+", dcTr', dA
T.P. Labeled with dGTI1 (however, LdCTl1 is 321),
Specific activity 4.4XI06cpa/nmola); 3oμg
{Rigo(dT>12~II1.80!l'-position trimyeloid leukemia virus 11L rice reverse transcriptase. After reacting at 43°C for 90 minutes, the Ik reaction was stopped with El)TA aqueous solution. Houenorukjljl;l;
The cDNA-mRNAΔ complex was separated using dk(1:I), precipitated with ethanol, and recovered. After the mRNA was decomposed and removed by treatment with alkali and 4λ, the synthesized j11 chain cDNA was precipitated with ethanol and recovered. The precipitation of this single-stranded cl)NA was carried out using the following reaction buffer i1'.
k was dissolved in 40 μl. Reaction buffer: 0.5mM dATI', dTTP, dGT+', (IC
TI'; 5mM Ml4CI2; 70mMKCI: 1.5
mM β-menolekabutr-9nol; 8 units E. coli DN
0 containing A polymerase I (Learn fragment/to)
．． IMI lepes buffer (pll 0.9). Double-stranded cDNA was synthesized by reacting at 15°C for 20 hours. The reaction was stopped by adding an aqueous solution of sodium dodecyl sulfate to the reaction mixture, and the double-stranded cDNA was extracted with a mixture of fanonolake and chloroform, precipitated with ethanol, and then recovered. The resulting double-stranded cDNA precipitate was treated with 50mM acetic acid ZnI-(pll4.5>.1mMZnSO4,20
Aqueous jlM1007l containing 0mM NaCI, 0.5% Chrycerol and 0.5 units of S1 Nuclea tF
/ and reacted at 37°C for 20 minutes to cleave the hairpin structure. The reaction was stopped by adding an aqueous LtEDTA solution, extracted with a phenol-chloroformo mixture,
Furthermore, after re-extracting with ether, cDNA was recovered by precipitation with ytanol. (3) Preparation of (IC)-tailed cDNA Add 100 Ill of the following silk formation reaction j1 buffer to the double-stranded cDNA obtained in step i, react at 37°C for 20 minutes, and double-stranded cDNA) to NA” Rigo (dC) tail to f. I added it. Reaction buffer: 1mM CoCI2, 0.1mM CoCl/
,0,2uybo')(A),O'. ImM3II-dc
Tr'' (specific activity fJ: 5400 (:Ill/pmol) and 130mM containing IO!It position terminal deA xynuclease tidyltranosferase: 130mM lithium dilate-30mMTris-11CIkA?+ritM
(pll6.8). The reaction was stopped by adding a 1ΣDTA aqueous solution, then extracted with a phenol-chloroform mixture, and then re-extracted with Coral.
A was precipitated with ethanol and recovered. This is 10m
MTris-11CI buffer iffl (pll7.4),
Dissolved in an aqueous solution containing 1mM TI) TA and 00mM NaC+ so that 0.2 llir of ``lico (dc) tailed cDNA per 11'' was dissolved [7]. (4) Oligo (dG) tail, 1 addition plasmid 1・p1
Preparation of mouth 322 DNA 20mM Tris-11Cfluenitk (pll7.
4). lOmMM14CI2.50mM(NH4)2S
n. per O4 and 11. +11. + no u/lilli1'
Water containing i Alfminwo dgi & IOOμI 1) Old <3
22 as lO/l+! ? After dissolving the mixture, 15 mononucleotides of restriction polypeptide PStI/donuclease (1:1) was added and the reaction was allowed to proceed at 37°C for 1 hour. After the reaction was completed, the reaction solution was extracted with phenol, and the DNA was recovered from the water by ethanol precipitation. The resulting DNA was mixed with 3II-dG instead of the aqueous solution (3II-dcTI) used for the above-mentioned λrigo(dC) tail addition.
Dissolve in 200 μl (containing TP) and add termylyl deoxynucleotide l'iQ111 {◇
Approximately 10 to 15 dG residues of ul were incorporated by reaction at 37° C. for 20 minutes. The reaction r'7 was eluted with a water-saturated fluoro-chloroform mixture, and the oligo(da) Boului monoadded plasmid pBR322 DNA was recovered from the aqueous layer by ethanol precipitation. This was dissolved in the same solution as for the oligo(C+C)-tailed cDNA so that each lsl contained 2 μg of the oligo(dC)-tailed zocysmid pllR322 DNA. (5) Production of I'll recombinant plasmid Alig (dC)
Add 50/ml of 1″-Louis I cDNA solution to Aligo (d
G) J-ru addition 1) l-R3221) 50 liters of NA solution
(120 minutes at 57°C for IO minutes at 5°C.
60 minutes at 45°C, 60 minutes at 35°C and 3
{Annealing was performed by incubating in a vortex for 60 minutes to prepare recombinant Bushimi lysed i1k. (6) Selection of transformants - Using the III recombinant plasmid solution obtained in Section 1, E. E. coli χ1776 strain was transformed. That is,
[．． coli χ1770K, Diaminobimeli 71! t
Absorbance ((!O
Onm > 0.5, the bacterial cells were collected using an F size centrifuge with a cooler, and 10mMT containing 50mMCaC+2 was added.
It was dispersed in ris-11CI buffer (pll7.3NOwl) and centrifuged again at 0°C to precipitate it.
Disperse in buffer 2-1 and incubate with static iF for 5 minutes at 0°C.
7L. This dispersion 0.21 was added with the above recombinant Zolami l'IFf.
Add and mix ilk011, leave at 0°C for 15 minutes,
After holding at 42°C for 2 minutes, the previous 18 tests were carried out once a month. ：Same as the one with the addition of ■, -bu ``'' 051 and 1
Time swing; 1. ;Mu゛7 nourishment was carried out. Take a part of this culture i1&, If! In addition to the ingredients of lI'l8, it was spread on a 1,1/Ross agar plate containing Tetler Iclino+51zlr/1@: about 12"+flffflIij blue at l7'C,
No 1 Razu Ikuri/Choose a laxative fungus (;I) NA Soi/
I made Nori a'1. (7) Hybridization. Regarding the cDNA library described in the test rules, a plasmid containing cDNA 8 encoding rabbit cancer necrosis r- was used as Il7.
32 pi protocol to screen transformants.
) Colony/1 hybridization using NA blu7-b
The F-N H+7 test was performed using the method of Llanahan et al. [Gen
e, 10.63 (+980)]. 32p
The NA probe was synthesized in step (2), using the mRNA obtained in step (1) as a template. However, 32rI-dCTP, which has a high release rate and a specific activity of 1, was used and was labeled at a high concentration. Through this test, negative transformants were selected that strongly bound to the induced positive probe and imitated a recombinant plasmid containing the IuM sequence that did not hybridize with the induced G.mylis probe. 50 out of about 20,000 colonies. 1 bite, 21?] X was taken out. These selected strains were then subjected to the t+lan test.
iaLD;,T. , atal, (cd) “MOIQc+
Ilarclon+ng”.329 (1980), Co
ldSpringllarborI. ab. , written in 4'
lO) It was performed according to the force method. Blasmid DNA was extracted from each transformant, heat-denatured and fixed on a nitrocellulose filter, and the above (II1)
Poly(
A) Add the mRNA fraction and react at 50°C for 30 minutes.
Three hybridization tests were performed. After elution and recovery of the bound mRNAΔ, injected into African frog egg 1li cells.
The recovered InRNA or rmRN from chronic cancer
It was tested whether it was A or not. This test revealed that 1! out of the 20 transformants selected above! sweetfish necrosis factor m
We found 3 strains containing 1cl)NA'a+ plasmids that strongly hybridized with RNA. Buchsumi F has the most unique cDNA (approximately 750 bP) among them.
Therefore, with the limited amount of alcohol, the NA cutout was obtained and used as a probe for secondary screening. This DN
The A fragment was labeled with 32p, and the cl obtained from (6) JII
) NA library was again subjected to the high solidization test, and transformants having a plasmid containing the labeled plasmid (cl)NA that strongly combined with J-p were selected. cl) Of approximately 60,000 colonies in the NA library, 98 were positive colonies. From these cl)
NA was excised with the restriction enzyme PstI, and its size was examined by polyacrylamide gel electrophoresis.
17 clones with 1 size were selected. Among these, the transformant containing the largest cDNA (cell number: RTNFR02, cloned DNA type: pR'
rNF802), the curnized DNA was amplified, and the base sequence was determined by the method described below. (811 (- Determination of the base sequence of -+-) DNA The strain (RTNF802) selected in section (7) was cultured in 1-gloss supplemented with diaminopimeli/acid and thymidine to obtain bacterial cells. The bacterial cells were collected from Wilkie et al.
le; cAC+clSRes. ,7.859(+979
)] to obtain busismid DNA. This plasmid 1) NA was digested with restriction enzyme Jr'stl and purified and separated to obtain 1) NA. This Ku [Fu 7
The DNA fragments were digested manually using restriction enzymes A, and the 1-base sequence of each commercially available restriction enzyme fragment was extracted using Mλxam.
-Gilbert method I) Determined from terminal labeling by delinooxidation 321), base-specific chemical decomposition reaction, gel electrophoresis, and 1F sigmography. In FIG. 3, arrows indicate the restriction tract cleavage site used in the salt sequence determination, the direction in which the base sequence was determined, and the ilii circle. two:
The part shown here represents the translation region of Eugi carcinoma necrosis factor. The base sequence is shown in Table 1. Nos. 1 to 15 are c
This is a G-interceptor tail added to incorporate DNAΔ into a vector. Numbers +6 to 276 are base sequences that are determined when ml of polypeptide necessary to construct the n11 precursor of cancer necrosis r is generated. No. 277-2 old number 11. iM coats the N-terminus of rabbit cancer necrosis with the amino acid sequence corresponding to 411, and the salt J1 of Nos. 715 to 738, 1-1 (is C
It coats the amino acid sequence of the terminal part (sequence enclosed by C==a in Table 1). Following the C wood terminal amino acid (0 isine), there is Tsuyoshi+l::+do7 (TGA). Annoying. 1. The number of amino acid residues translated from the base sequence (nos. 277 to 738) of the region coding for the polypeptide of cancer necrosis factor 1 is 154;
(, ilj. are as shown in Table 2. As is clear from Table 2, the purified Uri 1! cancer necrosis factor amino acid introduction and measurement molecule; and 7
They agreed within a constant error of 1111. In addition, the numbers in Kano:J in the table are the integer values obtained by rounding off the analysis values. Propio to a single purified tumor of cancer necrosis factor (body ir 12.5-3.0 kg)
50. nibacter u1aC:n(!S) killed cells were injected into the ear vein. Eight days later, 1007 liters of E. coli-derived Noriboli saccharite was injected into the ear vein. Two hours later, heart Th'. !J, whole blood was collected. After adding Hebari/sodium at +001-position per 1001 to the collected blood, a refrigerated centrifugation operation was performed at 5,000 rpm for 10 minutes to remove blood cells and insoluble solids. 400 birds. Plasma 24/24 with a titer of 3,000 units l1 was obtained from a rabbit.
After stirring for 1 hour, the pore size was 3 μm. 1μ and 0
．． It was sequentially filtered through a 2μ filter. After adding 0.04M, Tris-11CI buffer (pll7.8)+27 to the introduction i+1!241, 0. IMNa
0.04M Tris-TICI Woe impulse including CI
Gradually add f-t to a column (27 Next, empty! Equilibration buffer i1k75li and 0.15M NaC
0.04M Tris-11cIffl&i solution containing l(
pll7.8) 501-t? li next washing j'llffl,
0. I8MNa. 0.04MTriS-11 including CI
Solution 111 was carried out using cItJ buffer (pl+7.2). The eluate is 8/long ii! ＋il, and active jlii fractions were collected. The same volume of this active fraction fitO) 0.04MTr
After diluting by adding is-11cI buffer (pll7.8), it was injected into h lamb (foxI:lem) of January:AI7-Sephulose CL-On. Then, 0.IMN
0.04M Tris-1ICIf, Y”4 including aCI
Minami ink (pll7.8>l/wo811, +te wash jp(
, Ta (D, 0.04MTri including O.I8MNaCIt)
It was eluted using s-11c15 ill'(pll7.2>51).The eluate was fractionated through 25o1
Collected 1111 minutes. Next, this eluate was transferred to a container and immersed in a 70'C frying bath.
The eluate was heated without stirring until the temperature reached 41 degrees GO'C. Thereafter, the mixture was transferred to another CO'C frying bath, heated for 30 minutes, and then rapidly cooled to 4°C. The heat-treated solution was subjected to ultrafiltration to cause zero shrinkage. Including 0.1M NaC+. 005M')7mlm9
Sen 2 fully balanced with 4i1k' (pl17.4)
Kasim (5X8) of Kuril S-2QQ (Pharmacia)
4 fl of the concentrated iP described above was eluted with the same buffer solution. 40il each minute 1j) iL 7. ζV {Both parts were removed and reduced by +5 by ultra-σ2 passing. The concentrated solution of the active fraction obtained by gel filtration was
Added to 4-rate roast and 7-loin lamb. PoraLh, J. , atal. ,Nature,25
8.5 fl8 (+975) was filled with a column (+. ,
1? ,/-1 l zinc monoxide aqueous solution I201 was flowed. Then O. 0.05 Mυ acid buffer containing 1M NaCl
After sufficient equilibration with 11i (pl+74), the dark blue il moxibustion obtained in the previous step was mixed with {. The non-adsorbed fraction eluted with the same buffer was collected and collected. Most of the live V1 was recovered in both parts. Prepurification-1: Concentrate the active fraction subjected to iIJ in step 1. 0.
0.005M phosphate buffer (p
Toyobar IIW-5 which was fully modified at l+7.4>
5 (East〆Y-Soda Co., Ltd.) Kashi L, (15X9
0+.. ). It was eluted at the same buffer port and the active T+ fraction was collected. The yield of active V+ was 60%, and the degree of purification was 7.5x10' times using all purified I'l as iI+il-. The purified rabbit sample 7 thus obtained (the ratio of necrosis factor V1 was O.OX106 units/g protein),
1) Definition of 1 is based on Japanese Patent Application Laid-Open No. 1988-+7+3
It is the same as that of No. 3o). Also, move 4NL to the mouse
In the biological evaluation using the Math^ flesh, 71 when 3,000 to 5,000 units per animal was administered intravenously was also (+) or higher. Reference Example 2 Properties of ``v゛-+!IX+ Necrosis Factor (1) Molecule 111 Measurement Purified rabbit cancer necrosis factor obtained in Reference Example 1, T
High school 1 using SK-G3000SW color t, ('AI%n'! Co., Ltd.? J)! ! Molecule 1il was determined by liquid chromatography. The solvent was 0.2M phosphoric acid
ill (pll7) and the same buffer containing 8M urea and 0.5% β-mercabutoJ tanol were used. Molecule 1, Molecule II1 using Lfill standard standard protein
The gland was examined for f'1, and the molecule L1 was measured for serum albumin (1i.l'ix10').・Fri''one-s;j,;(fu, one toisomera-V(5
．． :+x10'). A-valbumin (4.5X10),
Pig Peb7no (1.27XIO'), Soybean Tribunno Inhibitor (2.05x10'), Horse Mikari Globino (1.78xIo').・Nma bu''kuul. C<I
．． 24x! 0') [The number I/1 in Kanov does not indicate the molecular weight (dalt/). As a result, the molecular weight of Urigi cancer necrosis factor was approximately 450,000 Daltons in the absence of urea, and approximately 160,000 Daltons in the presence of urea. (2) Wooligi cancer necrosis factor manufactured by Ami7 Milk Composition 1l1 was hydrolyzed with acid according to a conventional method, and then each amino acid was analyzed using the vIifk amino acid analysis system (Shimako Seisakusho) using fluorescence method using lutaldehyde. Quantitated. Purified Utsugi cancer necrosis prisoner 15~'
:IOttH in 6N hydrochloric acid at 110°C, 24,48.
Hydrolysis was carried out by heating for 72 hours. Contains 1i1 of each amino acid
{a'i is the average full1 at 33rd time and the correction value (Ser
, Thr, Val, I+ (!). The results are shown in Table 2 (n'liff). (;3) Amino acid sequence determination 1) The amino acid sequence of the N-terminal portion of purified carcinoma necrosis factor was determined by the Edmanolysis method [Arch. Old ochem. old oph
ys. , 22,475 (+9441)]. 1. After reacting 0.3 mg of Wooligi Cancer Necrosis Factor (manufactured by 1) with phenyl cyanate and decomposing it sequentially from the N-terminus, the free phenyl cyanide/toino amino acid derivative was treated with Zorbax ODS (4.flX250).
ml1. The amino acid sequence of the N-terminal portion was determined by high-performance liquid chromatography using Jubon Co., Ltd.). ii) The amino acid sequence of the C-terminal portion was determined by the Hidofuji 7 digestion method and the enzyme method using carbo, l-mbbutidase. Human trazine decomposition is Akahori's force rJ+ [Ilull. Che
Il. SocJap. , 25.214 (+952)], purified Ugi cancer necrotic convict 30ugwo4HH (IOO'C-('flII!7) was decomposed in 10oul of water-hytracine, and then treated with benzaldehyde to obtain the amino acid hydrazito derivative. Furthermore, purified rabbit tumor necrosis factor was treated with carbo1.pebutidase A and Y (
The ratio of enzyme and substrate is 1゛25 and l:'+000 respectively)
The amino acids generated in JF1iL were analyzed over time from 2 minutes to 180 minutes after the start of the reaction. As a result, it was revealed that the amino acid sequences of the N-terminal and C-terminal portions have the structure shown below. N-terminus: Se
r-8la-Ser-8rg181a...C-terminus:...Val-Tyr-I'he-Gly-1le-1
le-^la-Leu Sansho Example 3 Anti-Uri~1! Preparation of cancer necrosis antibody Purified rabbit cancer necrosis antibody obtained in Reference Example 1 1.9XIO
A solution containing ``!It'' was emulsified with Freund's complete adjuvant and injected subcutaneously at several points on the back of Mormosots.Then, 1, 3, and 6 weeks later, they were immunized in the same manner. Eight weeks later, the same purified rabbit cancer necrosis factor II1 was injected intraperitoneally twice along with aluminum hydroxide and gel. [f] weeks after the first immunization, whole hearts were harvested, and the serum was separated by centrifugation to obtain antiserum containing anti-rabbit cancer necrosis antibodies. This antiseptic solution was applied three times to a Cef71.1-S43 (Pharmaciane 1) column that had been adsorbed with normal rabbit serum components. Nonspecific antibodies were removed by passing through M4L to obtain a purified antiserum containing only specific antibodies against Wooligi cancer necrosis factor. This antiserum formed an r11- precipitation line only between purified rabbit cancer necrosis factor and purified rabbit cancer necrosis factor by immunoelectrophoresis and in-gel double diffusion. This purified antiserum was diluted approximately 60,000 times to obtain a 1.0-fold dilution of Ugi Island necrosis factor. - Ability to neutralize 1100 units of iI cytotoxic activity by 50%.

[Brief explanation of drawings]

Figure 1 shows Wooligi cancer necrosis factor mRN 8 acids + 1 urine l-γ
Fig. 2 is a diagram showing the results of gel electrophoresis, and Figure 2 shows the results of the injection of Aguinea carcinoma necrosis factor mRNA into the Aguinea carcinoma necrosis translated in Xenopus laevis oval cells. L-929 cytotoxic activity of factor is anti-Usa 1! It is a 'i:>ic-J diagram showing that cancer necrosis is neutralized by the antibody. :+-F-J cloned DNA (pRTNF802). (This indicates the position and size of the restriction enzyme cleavage site used for sequencing. -504 Handbook Supplement (self-prompted) November 1980. 30th Japan Patent Office Commissioner Shiga Gakudono 1) Display of the case 1982 Patent Application No. 228790 2 Name of the invention Cloned DNA that coats rabbit cancer necrosis factor 3 Person who makes corrections Related to the case Patent applicant Address 3-25 Doshomachi, Higashi-ku, Osaka Name 291 University Nippon Pharmaceutical Co., Ltd. '2:47'generation''Fk Fuji gN man'
f'゜972 Shasaka 4 Agent ~ Address 33-94 Enohonmachi, Suita City, Osaka Prefecture Dainippon Seiro Co., Ltd. r1' General Research Institute Name Kojima +: 727: I Kojima - Kojira Sayaen 5 Correction Contents of the amendment in column 6 of “Detailed Description of the Invention” of the subject Meikaku 1 (1) In line 7 of page 30 of the specification, “3o” was replaced with “3o”.
3” and correct it. (2) On page 36, line 6 of the specification, replace “3o” with l.
Correct with rl80j,

Claims (1)

[Scope of Claims] A DNA having a salt p sequence encoding a polypeptide containing the amino acid sequence of fl1 Woolly cancer necrosis factor, or containing an allelic mutation, degeneracy of the ill gene code, or modification of the - region. A variant of said DNA. f2] i1! Claim 1, wherein the salt μ sequence encoding rebebuzido is ai! 111 I) Variants of the DNA containing NA or allelic mutations, degeneracy of the genetic code or modification of the - region. (3) Code cheating on polybegyd? (r according to claim 1, whose sequence is) NA or allelic gene mutation, degeneracy or partial modification of the gene code
Mutants of NA. (4) Claim mIJJ1 in which the nucleotide sequence encoding the polypeptide is: A variant of said DNA. (5) The sequence iii. 1) Mutants of NA. (6) l) NA or pair) gamma gene mutation according to any one of claims 1 to 5 included in the vector. A variant of the DNA containing a shortening of the genetic code or a modification of the - region. (7) The vector is a gene expression vector capable of expressing a polypeptide containing the amino acid sequence of a horse necrosis. Coat IVi weight L< indicates a variant of the DNA containing some modification. (8) The amino acid sequence of rabbit cancer necrosis factor is 11. '3m1' Coat the base platter I1
)NA! ．． i is an allelic mutation; a host transformed with a vector DNA incorporating a variant of the DNA containing a truncation or modification of the - region of the n gene. (!) The host according to claim 8, wherein the vector is an expression vector capable of expressing a polypeptide containing the amino acid sequence of cancer necrosis factor. (jO) Special claim 8 which is a microorganism or cultured cell
Section 9 or Section 9 Host of the company.